Cinobufacini retards progression of pancreatic ductal adenocarcinoma through targeting YEATS2/TAK1/NF-κB axis.

BACKGROUND: Cinobufacini, a sterilized hot water extract of dried toad skin, had significant effect against several human cancers. However, there are few studies reporting the effect of cinobufacini on pancreatic cancer. PURPOSE: To investigate the effects of cinobufacini on the progress of pancreatic ductal adenocarcinoma and the underlying mechanisms. METHODS: Cell counting, EdU incorporation and flow Cytometry were performed to evaluate the effect of cinobufacini on cell cycle and growth. MIA-PaCa2 cells were implanted into the nude mice to determine whether cinobufacini represses PDAC progression in vivo. Luciferase reporter assay, western blotting and qPCR were carried out to measure the activity of NF-κB pathway and the alteration of YEATS2 and TAK1. Ectopic gene expression introduced by plasmids was used to verify the molecular mechanism. RESULTS: Our results showed that cinobufacini induced cell cycle arrest and inhibited the growth of PDAC cell in vitro, and repressed MIA-derived PDAC in vivo. Cinobufacini inhibited the phosphorylation of IKK, IκB and NF-κB p65 in PDAC cells. Furthermore, cinobufacini decreased the abundance of intracellular YEATS2 and total TAK1 protein in a time- and dose dependent manner. Ectopic expression of YEATS2 re-elevated the level of TAK1 and phosphorylated IKKα/ß, IκBα and p65 after cinobufacini treatment in PANC-1 cells. CONCLUSION: Cinobufacini retards the growth and progression of PDAC in vitro and in vivo through YEATS2/TAK1/NF-κB axis.

Amphiphilic Cationic Peptide-Coated PHA Nanosphere as an Efficient Vector for Multiple-Drug Delivery.

Amphiphilic core-shell (ACS) nanoparticles are gaining increasing research interest for multi-drug delivery in cancer therapy. In this work, a new cationic peptide-coated PHA nanosphere was prepared by self-assembly of a hydrophobic core of biodegradable poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and a hydrophilic shell of fusion proteins of PHA granule-associated protein (PhaP) and cationic peptide RALA through a strong hydrophobic effect. The hydrophobic drug curcumin (Cur) was encapsulated in PHBHHx nanoparticles. The chemotherapy drug 5-fluorouracil (5-FU) was administered in the form of its metabolite oligomeric 5-fluorodeoxyuridine (FUdR). Fifteen consecutive FUdR (FUdR15S) were adsorbed on the surface of PHBHHx nanoparticles by electrostatic interaction with RALA to form Cur@PHBX-PR/FUdR15S. Such amphiphilic cationic nanospheres had 88.3% EE of Cur and the drug loading of Cur and FUdR were 7.8% and 12.1%. The dual-drug-loaded nanospheres showed a time-differential release of Cur and FUdR. In addition, Cur@PHBX-PR/FUdR15S exhibited excellent anticancer activity and played a vital role in promoting the synergistic effect of FUdR and Cur in gastric cancer cells. The exploration of antitumor mechanisms demonstrated that Cur improved the activity of apoptosis-related proteins and cancer cells sensitized to FUdR. This amphiphilic core-shell system can serve as a general platform for sequential delivery of multiple drugs to treat several cancer cells.

Macrophages Loaded with Fe Nanoparticles for Enhanced Photothermal Ablation of Tumors.

Magnetic iron nanoparticle-based theranostics agents have attracted much attention due to their good magnetism and biocompatibility. However, efficiently enriching tumors with iron nanoparticles to enhance the treatment effect remains a pressing challenge. Herein, based on the targeting and high phagocytosis of macrophages, an Fe nanoparticle-loaded macrophage delivery system was designed and constructed to efficiently deliver iron nanoparticles to tumors. Hydrophilic Fe@Fe3O4 nanoparticles with a core-shell structure were synthesized by pyrolysis and ligand exchange strategy. Subsequently, they were loaded into macrophages (RAW264.7 cells) using a co-incubation method. After loading into RAW264.7, the photothermal performance of Fe@Fe3O4 nanoparticles were significantly enhanced. In addition, Fe@Fe3O4 nanoparticles loaded into the macrophage RAW264.7 (Fe@Fe3O4@RAW) exhibited a good T2-weighted MRI contrast effect and clear tumor imaging in vivo due to the tumor targeting tendency of macrophages. More importantly, after being intravenously injected with Fe@Fe3O4@RAW and subjected to laser irradiation, the tumor growth was effectively inhibited, indicating that macrophage loading could enhance the tumor photothermal ablation ability of Fe@Fe3O4. The macrophage mediated delivery strategy for Fe@Fe3O4 nanoparticles was able to enhance the treatment effect, and has great potential in tumor theranostics.

Hesperetin, a SIRT1 activator, inhibits hepatic inflammation via AMPK/CREB pathway.

Silent mating type information regulation 2 homolog 1 (SIRT1) is an important inflammatory regulator, which epigenetically reprograms inflammation by altering the acetylation of NF-κB. Hesperetin, as a common flavonoid, has been proven to have a significant effect on acute inflammatory diseases. However, the detailed molecular mechanism by which hesperetin alleviates inflammatory response and accompanied tissue injury is poorly understood. Our results show that SIRT1 is required for the inhibitory effect of hesperetin on inflammation. Hesperetin suppresses the acetylation of RelA/p65 to reduce NF-κB activity by inducing SIRT1 expression. Mechanistically, hesperetin increases SIRT1 expression through AMPK/CREB pathway. Additionally, the protective effect of hesperetin against LPS/D-GalN-induced hepatitis in mice is also dependent on SIRT1. Our study suggests that hesperetin is an SIRT1 activator and could be potential candidates for the treatments of inflammatory conditions.

Neohesperidin enhances PGC-1α-mediated mitochondrial biogenesis and alleviates hepatic steatosis in high fat diet fed mice.

BACKGROUNDS: Mitochondria plays a critical role in the development and pathogenesis of nonalcoholic fatty liver disease (NAFLD). Neohesperidin (NHP) could lower blood glucose and prevent obesity in mice. However, the direct effect of NHP on hepatic steatosis has not been reported. METHODS: Mice were fed with either a chow diet or HFD with or without oral gavage of NHP for 12 weeks. A variety of biochemical and histological indicators were examined. In vitro cell culture model was utilized to demonstrate underlying molecular mechanism of the effect induced by NHP treatment. RESULTS: NHP increases mitochondrial biogenesis, improves hepatic steatosis and systematic insulin resistance in high fat diet (HFD) fed mice. NHP elevates hepatic mitochondrial biogenesis and fatty acid oxidation by increasing PGC-1α expression. Mechanistically, the activation of AMP-activated protein kinase (AMPK) is involved in NHP induced PGC-1α expression. CONCLUSIONS: PGC-1α-mediated mitochondrial biogenesis plays a vital role in the mitigation of hepatic steatosis treated by NHP. Our result suggests that NHP is a good candidate to be dietary supplement for the auxiliary treatment of NAFLD.

Cinobufacini Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice through Inhibiting M1 Macrophage Polarization.

Cinobufacini is a traditional Chinese medicine used clinically that has antitumor and anti-inflammatory effects. It improves colitis outcomes in the clinical setting, but the mechanism underlying its function yet to be uncovered. We investigated the protective effects and mechanisms of cinobufacini on colitis using a dextran sulfate sodium (DSS)-induced colitis mouse model, mainly focusing on the impact of macrophage polarization. Our results showed that cinobufacini dramatically ameliorated DSS-induced colitis in mice. Cinobufacini treatment reduced the infiltration of activated F4/80+ and/or CD68+ macrophages into the colon in DSS-induced colitis mice. More importantly, cinobufacini significantly decreased the quantity of M1 macrophages and the expression of proinflammatory cytokines such as interleukin-6, tumor necrosis factor α, and inducible nitric oxide synthase. Cinobufacini also increased the population of M2 macrophages and the expression of anti-inflammatory factors such as interleukin-10 and arginase-1 in DSS-induced colitis mice. Furthermore, our study demonstrated that cinobufacini inhibited M1 macrophage polarization in lipopolysaccharide-induced RAW 264.7 cells. Mechanistically, our in vivo and in vitro results showed that cinobufacini inhibition of M1 macrophage polarization may be associated with the suppression of nuclear factor κB activation. Our study suggests that cinobufacini could ameliorate DSS-induced colitis in mice by inhibiting M1 macrophage polarization.

NF-кB increases LPS-mediated procalcitonin production in human hepatocytes.

For years, procalcitonin (PCT) has been employed as a diagnostic biomarker for the severity of sepsis and septic shock, as well as for guiding the application of antibiotics. However, the molecular/cellular basis for the regulation of PCT production is not fully understood. In this study, we identified the signalling pathway by which the expression of PCT was induced by lipopolysaccharide in human hepatocytes at the mRNA and protein levels. This expression was dependent on nuclear transcription factor κB (NF-κB), as indicated by a NF-κB binding site (nt -53 to -44) found in the PCT promoter region. We also showed that microRNA-513b (miR-513b) was also able to bind to the 3'-untranslated region (UTR) of the PCT promoter sequence. Meanwhile, the activation of NF-κB down-regulated the expression of miR-513b. In conclusion, we suggest that NF-κB is capable of enhancing the expression of PCT by either directly activating the transcription of the PCT gene or indirectly modulating the expression of its regulatory component, miR-513b. Our results indicate a molecular mechanism responsible for the regulation of PCT production.

Lipin-1 determines lung cancer cell survival and chemotherapy sensitivity by regulation of endoplasmic reticulum homeostasis and autophagy.

Cancer cells undergo comprehensive metabolic reprogramming to meet the increased requirements of energy and building blocks for proliferation. Lipin-1, a phosphatidic acid phosphatase converting phosphatidic acid (PA) to diacylglycerol (DAG), is upregulated in lung adenocarcinoma (LUAD) cell lines and tumor tissues. In this study, we reveal high lipin-1 expression is correlated with poor prognosis of patients with LUAD. Knockdown of lipin-1 decreases cell viability and proliferation in LUAD cells, whereas it has less effect on nontumorigenic lung cells. Autophagy and ER stress play important roles in tumor initiation and progression. Lipin-1 knockdown induces the initiation of autophagy while disrupts formation of autolysosome. Lipin-1 silencing induces the activation of ER stress through the IRE1α pathway. Furthermore, we demonstrate disrupted ER homeostasis contributes to the cell phenotype, and the elevated autophagy initiation is due to the ER stress in part. For the first time, we show lack of lipin-1 enhances the sensitivity of LUAD cells to cisplatin treatment. Our results suggest that lipin-1 is a potential target, alone or combined with other treatment, for lung cancer therapy.

SLC2A5 promotes lung adenocarcinoma cell growth and metastasis by enhancing fructose utilization.

The metabolism of cancer cells is highly plastic. Cancer cells can change their preference for nutrient uptake under nutrient stress. Fructose is one of the most common carbohydrates in diet and its metabolism is also involved in the development and progression of tumors. GLUT5, encoded by SLC2A5, is the specific fructose transporter in mammalian cells. In this study, we found that SLC2A5 is significantly upregulated in lung adenocarcinoma (LUAD) patients and overexpression of SLC2A5 is highly correlated with poor prognosis of LUAD patients. The expression of SLC2A5 determined fructose uptake and utilization efficacy in LUAD cells. GLUT5 is critical for the survival of LUAD cells in fructose-containing culture medium. Depletion of SLC2A5 undermined cell proliferation and invasion meanwhile increased cell apoptosis. Overexpression of SLC2A5 enhances cell proliferation, migration, invasion, and tumorigenic. Compared to glucose, fructose is prone to strengthen intracellular-free fatty acid accumulation and ATP production. Moreover, inhibition of GLUT5 by specific small chemical inhibitor sensitizes LUAD cells to paclitaxel treatment. Taken together, our results suggest that GLUT5 could be a potential target alone or combination with other treatment for lung cancer therapy.

Astilbin ameliorates cisplatin-induced nephrotoxicity through reducing oxidative stress and inflammation.

Oxidative stress and inflammation are considered to be the main pathogenesis of cisplatin nephrotoxicity. Astilbin, a flavonoid with anti-oxidation and anti-inflammation function, has been used to treat heavy metal induced kidney injury. In this study, we investigated the protective effects of astilbin on cisplatin-induced nephrotoxicity and its underlying mechanisms. Our results showed that astilbin markedly inhibited cisplatin-induced cell apoptosis and recovered cell growth. Astilbin significantly decreased reactive oxygen species (ROS) accumulation and alleviated ROS-induced activation of p53, MAPKs and AKT signaling cascades, which in turn attenuated cisplatin-induced HEK-293 cell apoptosis. Astilbin effectively enhanced NRF2 activation and transcription of its targeting antioxidant genes to reduce ROS accumulation in cisplatin-induced HEK-293 cells. Furthermore, we found that astilbin obviously suppressed tumor necrosis factor alpha (TNF-α) expression and NF-κB activation, and also inhibited the expression of induced nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Finally, we confirmed that the effect of astilbin to improve renal oxidative stress and inflammation in cisplatin induced acute nephrotoxic mice. In conclusion, our study suggests that astilbin could ameliorate the cisplatin-induced nephrotoxicity by reducing oxidative stress and inflammation.

Polo-like kinase 3 (PLK3) mediates the clearance of the accumulated PrP mutants transiently expressed in cultured cells and pathogenic PrP(Sc) in prion infected cell line via protein interaction.

Polo-like kinases (PLKs) family has long been known to be critical for cell cycle and recent studies have pointed to new dimensions of PLKs function in the nervous system. Our previous study has verified that the levels of PLK3 in the brain are severely downregulated in prion-related diseases. However, the associations of PLKs with prion protein remain unclear. In the present study, we confirmed that PrP protein constitutively interacts with PLK3 as determined by both in vitro and in vivo assays. Both the kinase domain and polo-box domain of PLK3 were proved to bind PrP proteins expressed in mammalian cell lines. Overexpression of PLK3 did not affect the level of wild-type PrP, but significantly decreased the levels of the mutated PrPs in cultured cells. The kinase domain appeared to be responsible for the clearance of abnormally aggregated PrPs, but this function seemed to be independent of its kinase activity. RNA-mediated knockdown of PLK3 obviously aggravated the accumulation of cytosolic PrPs. Moreover, PLK3 overexpression in a scrapie infected cell line caused notable reduce of PrP(Sc) level in a dose-dependent manner, but had minimal effect on the expression of PrP(C) in its normal partner cell line. Our findings here confirmed the molecular interaction between PLK3 and PrP and outlined the regulatory activity of PLK3 on the degradation of abnormal PrPs, even its pathogenic isoform PrP(Sc). We, therefore, assume that the recovery of PLK3 in the early stage of prion infection may be helpful to prevent the toxic accumulation of PrP(Sc) in the brain tissues.

Remarkable reductions of PAKs in the brain tissues of scrapie-infected rodent possibly linked closely with neuron loss.

Prion diseases are irreversible progressive neurodegenerative diseases characterized in the brain by PrP(Sc) deposits, neuronal degeneration, gliosis and by cognitive, behavioral and physical impairments, leading to severe incapacity and inevitable death. Proteins of the p21-activated kinase (PAK) family are noted for roles in gene transcription, cytoskeletal dynamics, cell cycle progression and survival signaling. In the present study, we aimed to identify the potential roles of PAKs during prion infection, utilizing the brains of scrapie agent-infected hamsters. Western blots and immunohistochemical assays showed that brain levels of PAK3 and PAK1, as well as their upstream activator Rac/cdc42 and downstream substrate Raf1, were remarkably reduced at terminal stage. Double-stained immunofluorescent assay demonstrated that PAK3 was expressed mainly in neurons. Dynamic analyses of the brain samples collected at the different time points during the incubation period illustrated successive decreases of PAK3, PAK1 and Raf1, especially phosphor Raf1, which correlated well with neuron loss. Rac/cdc42 in the brain tissues increased at early stage and reached to the top at mid-late stage, but diminished at final stage. Unlike the alteration of PAKs in vivo, PAK3 and PAK1, as well as Rac/cdc42 and Raf1 in the prion-infected cell line SMB-S15 remained unchanged compared with those of its normal cell line SMB-PS. Our data here indicate that the functions of PAKs and their associated signaling pathways are seriously affected in the brains of prion disease, which appear to associate closely with the extensive neuron loss.